miR‐21 modification enhances the performance of adipose tissue‐derived mesenchymal stem cells for counteracting urethral stricture formation

Abstract The treatment of complicated long segment strictures remains to a challenge, and the substitution urethroplasty treatment is often accompanied by subsequent tissue fibrosis and secondary stricture formation. In situ injection of human adipose tissue‐derived stem cells (hADSC) could potential be applied for prevention of urethral fibrosis, but the cells transplantation alone may be insufficient because of the complicated histopathological micro‐environmental changes in the injury site. This study investigated whether miR‐21 modification can improve the therapeutic efficacy of ADSCs against urethral fibrosis to limit urethral stricture recurrence. MiR‐21‐modified ADSCs (miR‐21) were constructed via lentivirus‐mediated transfer of pre‐miR‐21 and GFP reporter gene. In vitro results suggested that miR‐21 modification can increase the angiogenesis genes expression of ADSCs and enhance its anti‐oxidative effects against reactive oxygen species (ROS) damage. In vivo results showed that miR‐21 modification contributes to increased urodynamic parameters and better formation of the epithelium and the muscle layer as compared to ADSCs transplantation alone groups. The results demonstrated that miR‐21 modification in ADSCs could improve urethral wound healing microenvironment, enhance stem cell survival through ROS scavenging and promote the neovascularization via regulating angiogenic genes expression, which eventually increase the ADSCs' therapeutic potential for urethral wound healing.

healing process after urethroplasty, which make it a more complicated issue for success management of urethral stricture disease.
Although the administration of anti-fibrotic drugs like halofuginone has been proved to limit recurrence of urethral stricture, none of these medicines was observed with sufficient therapeutic benefit. 6 Recently various strategies, including growth factors administration, gene therapy, and stem cell transplantation, have been used to improve the treatment of urethral stricture. 4,[7][8][9] Among these, the transplantation of mesenchymal stem cells (MSC)-the multipotent stromal progenitor cells-has been proved to enhance the tissue repair process and resulted in better performance. 10,11 Large number of papers indicated that both multi-potential ability to differentiate into specialized cells for injury replacement and its paracrine effects to regulate the wound healing process, contribute to high therapeutic potential of MSCs during tissue repair or regeneration process. [7][8][9] However, because of the unfavourable microenvironment in the repair site, such as inflammation and over-production of ROS induced oxidative stress, the poor viability of MSC at the transplanted site might significantly decreased its therapeutic potential performance. 12,13 It is important to improve the survival of transplanted MSCs and enhance the biological functions in vivo. Recently, many studies have tried to increase the repair performance of transplanted MSCs via various intervention methods, including gene modification and biomaterials-based tissue engineering approaches. In particular, increasing evidences proved that microRNAs (miRNAs)based gene modification of MSCs is proved to have great potential for regenerative medicine. [14][15][16] The evolutionary conserved miRNAs with length of approximately 20-24 nucleotide played important and various roles in the control of genes expression by negatively regulating the translation of target genes expression. 17,18 As so far, miRNAs are proved to be implicated in many processes of biology metabolism, including the cellular proliferation and apoptosis, neuronal patterning and tumorigenesis, etc. What's more important, the miRNAs also evolved or directly regulated many complicated cell fate decisions and diseases via interfering a large number of regulated genes.
Among these, the miR-21 was reported with multi-faceted regulation effects, especially in stem cell biology. Emerging studies indicated that miR-21 might contribute to the self-renewal, lineage differentiation and paracrine effects of MSCs. [19][20][21][22] Recently studies have also shown that microRNA-21 may play pivotal roles in the regulation of a variety of skin fibrosis, including keloid, and hypertrophic scar. 20,23 These studies indicated the potential application of miRNA-21 modification for enhanced MSC-based (therapeutic potential) regenerative purpose in the counteracting of urethral stricture formation.
To address this issue, we hypothesize that the miR-21 modification of human adipose-derived mesenchymal stem cells (ADSCs) might be able to enhance its therapeutic potential for the healing and reconstitution of the urethra postoperatively to limit urethral stricture recurrence. The lentiviral-based transfection was used to enhance the expression of miR-21, and the therapeutic potential of MSCs was also evaluated in vitro and in vivo as well.

| Lentiviral based transfection of ADSCs with miR-21
The miR-21 lentiviral vector was generated by sub-cloning the precursor miR-21 fragment into the Pac1/Nhe1 cloning sites of the plasmid FUGW, a self-inactivating, replication incompetent lentiviral vector that carries the human ubiquitin-C promoter driving an enhanced green fluorescent protein (GFP) reporter gene. Human embryonic kidney 293T cells were transfected using SuperFect Transfection Reagent (Qiagen, Hilden, Germany) with the expression system vectors. After that, the virions were isolated by ultracentrifugation of filtered supernatant and applied for transfection (Lenti-miR-21 group). The vector without the miR-21 insert was used as negative group (Lenti-GFP group).
Adipose-derived mesenchymal stem cells were transfected with lentiviral at third passage (P3). The efficiency of lentiviral gene transfer in ADSCs was optimize, and the highest transfection efficiency was applied for the following experiments. After 72 hours, the expression of GFP marker gene was observed using an Olympas inverted microscope system (Olympus IX71, Olympus, Tokyo, Japan), and flow cytometry to indicate the transfection efficiency.
The cell viability at firth, second and third day after transfection was measured by a Cell Counting Kit-8 kit (CCK-8) according to manufacturer's instructions.

| qRT-PCR analysis
Seventy-two hours after transfection, total RNA of the lentiviral vector transfected ADSCs was extracted using a total RNA pure Kit  Table S1.
The PCR was performed with a hot start at 95.0°C for 45 seconds, followed by 40 cycles at 95.0°C for 5 seconds and 60.0°C for 30 seconds. The experiments were repeated for three times.

| Western blotting
Seventy-two hours fter transfection, the cells were lysed in Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA) and the total proteins concentrations were quantitative measured using the BCA Protein Assay Kit (Beyotime, Beijing, China). Equal amounts (80 μg) of total proteins were loaded on a 12% SDS-polyacrylamide gel and separated by electrophoresis. After being transferred to a PVDF membrane, the sample was blocked with 5% defatted milk and followed by primary antibody incubation overnight at 4°C. The primary antibodies hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor-1 (HGF-1), stem cell factor (SCF), stromal cell-  Table S1.

| In vivo transplantation of ADSCs for US model
The Sprague-Dawley rats were applied, and all animal experiments were conducted with the approval of the Ethics Committee of Animal Experiments of Beijing, China. The rats were anesthetized with pentobarbital sodium at a dose of 30 mg/kg and randomly divided into four groups. The rats received a methylene blue marked catheter and blood flow was blocked using elastic tourniquet. The ventral penile skin was exposed and 100 μL of saline (US group) or ADSCs suspension (1 × 10 6 ) was injected into the urethral wall at four different sites semi-circumferentially along 0.8-1.0 cm of the exposed urethra with a 30G needle. 5-10 minutes after the injections, the rats underwent four partial incisions of the penile urethra with a 23G needle the rats. All layers of the urethral wall were surgical cut to visualize the catheter. After removing the catheter, the penile skin was sutured up with absorbable sutures. The rats injected with cell suspension of MSCs, the MSCs transfected with only marker genes GFP lentiviral vector and the MSCs transfected with miR-21 lentiviral vector were denoted as MSC, GFP and miR-21 groups, respectively (n = 12).

| In vivo functional and histological examination
Four weeks later, urodynamic parameters were measured. Briefly, the rats were firstly anaesthetized with chloral hydrate (350 mg/kg) and held under partial restraint in a restraining device. The bladder was catheterized through urethra by polyethylene catheter (0.7 mm outer diameter, 0.4 mm internal diameter). The catheter was to urodynamic testing machine and infusion pump via a T-tube, and the conscious rats underwent cytometry for micturition volume (mL) and maximum flow pressure (cm-H 2 O) parameters. To further measure the urethral diameter, the micro-ultrasound was performed using a Vevo 2100 micro-ultrasound imaging system (Visual-Sonics Inc., Toronto, ON, Canada) with a 40 MHz linear-array transducer (MS-550D). The urethral diameter was calculated accordingly at the injury site and six rats were included for each group.
The rats were then immediately euthanized to obtain the bladder, penile and urethral tissues for histological examination and Western blotting. As for histological examination, the tissues were dehydrated, cleared and embedded in paraffin as normal. Three-to five-micrometer slices were obtained for Masson's trichrome and von Willebrand Factor (vWF) staining. Images were observed with Olympus IX71 inverted microscope.

| In vivo RT-PCR and Western blotting
To analyse the mRNA expression of angiogenic relative genes in transplanted human ADSCs, total RNA of tissue was extracted and the expression of angiogenesis related human genes was detected by quantitative RT-PCR as described previously in 2.3. To measure the proteins extents, the tissues were lysed in Laemmli Sample FENG ET AL.

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Buffer (Bio-Rad) and the total proteins obtained. The proteins extents of HIF-1α, VEGF, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), Collagen III, Elastin and β-Actin (Cell Signaling Technology) were detected as described previously in 2.4, and the band intensity was normalized with β-Actin as the endogenous control.

| Statistical analysis
All data were expressed a mean ± SD (standard error). One-way ANOVA followed by the Student-Newman-Keuls test for post hoc comparisons were used to evaluate the statistical significance with P-values of 0.05 and 0.01. Statistical analysis was performed using SPSS Statistics (version 19.0, IBM Co., Chicago, IL).

| miR-21 increase the angiogenesis genes expression of ADSCs
The flow cytometry was performed to characterize the expression of the cellular surface proteins. As shown in Figure 1A, the cells after three passages mostly expressed MSC markers CD105 and CD90, but were negative for heamatopoietic-specific marker CD45. After being transfected with lentiviral vector, most of the ADSCs (over 90%) were observed with GFP expression, which was further confirmed by flow cytometry (Figure 1B,C). CCK-8 results suggested that modification of the ADSCs via lentiviral transfection has no significant impact on the cell viability of ADSCs.
In order to study the regulation effects of miR-21 on the angiogenic capacity of ADSCs, the angiogenic relative proteins expressions were evaluated using qRT-PCR and Western blotting. As shown in Figure 2, as compared to the ADSCs in normal group and Lenti-GFP group, both qRT-PCR and Western blotting results showed that the expression levels of angiogenic factors HIF-1α, VEGF, bFGF, HGF-1, SCF and SDF-1a were significantly increased in miR-21 transfected ADSCs in Lenti-miR-21 group (P < 0.05 or P < 0.01).

| Functional evaluation results
Four weeks later, the micturition volume and maximum pressure were performed to measure the urodynamic parameters of the rats with urethral stricture. As shown in Figure 4A,B, the results showed that micturition volume of MSC and GFP groups were higher than that of US group, and the mi-R21 group was observed with highest F I G U R E . 2 A, qRT-PCR and (B) Western blotting evaluation of angiogenic relative proteins extents and mRNA expression. *P < 0.05, **P < 0.01 volume. Maximum pressure results showed that MSC, GFP and miR-21 groups significantly decreased the pressure as compared to US group (P < 0.05 or P < 0.01).
The histological analyses were also performed to evaluate the safety of ADSCs transplantation and wound healing of the urethral tissue ( Figure 4C). According to macroscopic observation and

| DISCUSSION
Studies have shown that MSC transplantation could enhance the healing and reconstitution of the urethra tissue to counteract US formation. Genetic modification of ADSCs with therapeutic genes was also proved to increase the therapeutic potential of the stem cells. 7 The microRNA-21 plays critical roles in the regulation of a variety of skin fibrosis, which hold great potential for new diagnostic and therapeutic options for wound remodelling and maturation. 12,15,22 The present study demonstrates that miR-21 modification in ADSCs via lentiviral transfection improved the therapeutic potential of MSCs for urethral wound healing. Furthermore, we also F I G U R E 5 Micro-ultrasound observation and detection of urethral diameter. A, Representative microultrasound images of rat penile urethras from different groups, and measurement of urethral diameter accordingly 4 wk after injections (n = 6), *P < 0.05, **P < 0.01 F I G U R E 6 In vivo RT-PCR and Western blotting analysis results. A, In vivo RT-PCR measurement of the angiogenic relative genes expressions; (B) representative chemiluminescence images of blotted membranes and (C) summarized protein expression levels for HIF-1a, VEGF, iNOS, eNOS, collagen III and elastin accordingly. *P < 0.05, **P < 0.01 investigated the potential underlying mechanism involved during the healing process, with results suggesting high levels of angiogenic factors expression for accelerated vascular maturation and favourable collagen remodelling.
Urethral wound healing after injury is a complex biological process. 5 The application of MSCs has proved to be able to improve wound healing via cell differentiation and paracrine effects. However, unfavourable microenvironment factors caused multiple impairments in cellular responses would hinder wound healing process. Among these factors contributing to impaired wound healing or excessive scar formation, the impaired production of cytokines, inflammation, oxidative stress and reduced angiogenesis are crucial. Sufficient vascularization of injury tissue is imperative for adequate wound healing and remodelling. In addition to exchange of the nutrients, oxygen and growth factors, etc., blood vessels were also proved to regulate tissue/organ growth, and repair by communicating with targeted tissues via its own angiocrine signals.
Increasing studies also indicated that MSCs derived angiogenic proteins could regulate the neoangiogenesis injury tissue for promoted wound healing process. Recently study also proved that miR-21 overexpressed MSCs can effectively induce angiogenesis potential via promoting HIF-1a activity. 24 In the present study, in order to understand the mechanism of miR-21 modified ADSCs in counteract urethral stricture formation, we examined the expres- Together, these results suggested that ADSCs transplantation could effectively improve the healing and reconstitution of the urethra tissue to counteract US formation, and the miR-21 modification via lentiviral transfection can increase the therapeutic efficacy.

| Limitations of this study
Despite these encouraging results, there are still some limitations in the present study. Although our primary data proved that miR-21 modification of ADSCs could increase the ADSCs' therapeutic potential for counteracting urethral stricture formation, the long-term therapeutic effects of it need to be further verified. What's more, the main purpose of this study was to test the feasibility of miR-21 modification for enhanced MSCs therapeutic potential for the healing and reconstitution of the urethra postoperatively, which could potentially be used to limit urethral stricture recurrence after urethrotomy. However, if local injection of ADSC, especially for gene modified ADSCs, could be used for treatment of urethral stricture that had been formed still need to be tested. What's more, although our results indicated the regulation effects of miR-21 modified ADSC on stem cell survival, ROS scavenging and angiogenesis, it is still insufficient to completely understand the whole roles of ADSCs counteracting urethral stricture formation, as well as the underlying cellular and molecular mechanism. Because urethral wound healing after injury is a complex biological process and involved many other cell types, such as inflammatory response and relative cells. The immunomodulatory benefits of miR-21 modification on ADSC as well as the molecular mechanism or signalling pathways involved in it is under investigation.

| CONCLUSION
The present study demonstrates that miR-21 modification in ADSCs could improve urethral wound healing microenvironment, enhance stem cell survival through ROS scavenging and promote the neovascularization via regulating angiogenic genes expression, which eventually increase the ADSCs' therapeutic potential for urethral wound healing. The current findings may offer new wide-reaching implications for the future of substitution urethroplasty and overcome recurrence of strictures after urethrotomy.

CONFLI CT OF INTEREST
The authors confirm that there is no conflict of interests.